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High Efficiency Transformation 2.0
High-Efficiency Yeast Transformation 2.0 This is the easiest high-efficiency method I’ve found (from C. Boone’s website). It’s ideal for integrating nutritional or drug-resistance markers as well as auxotrophy markers, in place of any genomic locus you’d care to delete. When only a couple of transformations per parent strain are being done, can transform in the morning since dilutions of an overnight YPD culture can be done when the overnight is started (e.g., 4 ml of inoculated YPD can be diluted 5x, 25x, 125x and 625x). In the morning, the culture that is closest to an OD of 0.5 to 1 is used and rest discarded. Do not discard the 1ml you use to measure the OD. You need that for the transformations. When doing more than a couple of transformations per parent strain, 25 ml or more of YPD in a standard 125 ml Wheaton bottle is inoculated with 2 ml late log overnight culture (make several dilutions of the 2 ml culture when it is started and discard the ones you don’t need in the morning). Pre-Steps 0.5. Make sure that the 95C and 42C heat blocks are turned on and heated to the correct temperature before beginning. 0.8. Remember to take the time to think about your controls before you begin (like a mock transformation… hint hint wink wink) 1 LiOAc + CARRIER DNA: For each 10 transformations add 0.3 ml 0.1 M LiOAc to 30ul (freshly boiled using 95C heat block for 15 minutes and then bring to room temp using ice) 10 mg/ml salmon sperm DNA. 2 GOOP: For each 10 transformations freshly mix 0.4 ml 0.5 M LiOAC and 1.6 ml Boone PEG (Boone PEG: dissolve 150 gm PEG3550 in 165 ml ddwater and filter sterilize). PEG3550 is in chemical shelves. For EACH transformation... 1) Pellet yeast from 3 ml of a mid-log culture at 3000 rpm for 2 min in a 15ml conical tube in the tabletop fuge swinging bucket rotor. 2) Aspirate supernatant. Re-suspend in 3 ml 0.1 M LiOAc and re-spin at 3000 rpm for 2 min in a 15ml conical tube in the tabletop fuge swinging bucket rotor 3) Aspirate Supernatant. Re-suspend in 1 ml 0.1 M LiOAc, transfer to a microfuge tube, and re-spin in a microfuge tube at 3000 rpm for 2 min in the microfuge. 4) Aspirate using P1000. Re-suspend in 30 ul 0.1 M LiOAc + Carrier DNA (Boiled Salmon Sperm DNA). 5) Add up to 15 ul DNA (e.g., PCR product that had been purified using a Qiaquick column). 6) Sit 15 min. at room temp. 7) Add 150 ul Goop (can vortex to mix) 8) Sit 30 min at RT. 9) Incubate in a 42 water bath for 15 min. 10) Spin yeast out slowly (2000 rpm, 2 min). 11) Aspirate supernatant and re-suspend yeast in 0.5 ml YPD. 12) Leave at 30C (incubator, not shaker) for 1 hr (or 4 hr if the phenotypic lag of a drug-resistance marker needs to be overcome).. 13) Gently re-spin at 2000 rpm for 2 min , aspirate out the YPD. Then, re-suspend in 120 ul of water… put on selective plate (LOOK BELOW) - IF USING KANMX/HYGROMX/NATMX TO MAKE DOUBLE DELETION FROM A SINGLE THAT ALREADY HAS ONE OF THE THREE DRUG MARKERS, USE SELECTION PLATES WITH BOTH MARKERS IN THEM. MARKERS ARE KNOWN TO SWITCH AT THE SAME SITE RATHER THAN TWO DIFFERENT LOCI.